Several publications are referenced in this application by author name and year of publication in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
The degradation of cellular proteins is necessary for the biological well-being of all organisms. Regulators of cell growth and development, and components of the immune and cellular defense mechanisms are regulated by proteolysis. Membrane receptors and transcription factors activated by cytokines, such as interleukins and interferons, are regulated by protein degradation.
The major pathway of intracellular proteolysis involves the ubiquitin/proteasome system. Ubiquitin, a 76 amino acid polypeptide, is the most highly conserved protein in eukaryotic evolution. There are only 3 amino acid differences between yeast and human ubiquitins. Extensive studies during the past decade have shown that the covalent attachment of ubiquitin to cellular proteins marks them for destruction. Substrates that are linked to ubiquitin are degraded by a multicatalytic protease called the proteasome. During the past few years many targets of the ubiquitin/proteasome system have been discovered and remarkably they include a broad range of regulators of cell growth. Some of the proteins destroyed by the ubiquitin/proteasome system include cyclins, cyclin-dependent kinases (CDK's), NFkB, IkB.alpha., cystic fibrosis transduction receptor, p53, ornithine decarboxylase (ODC), 7-membrane spanning receptors, Cdc25 (phosphotyrosine phosphatase), Rb, G.alpha., c-Jun and c-Fos.
The ubiquitin/proteasome pathway is also essential for the stress-response and for the generation of antigenic peptides in MHC Class I molecules. It is clear that defects in the functioning of the ubiquitin/proteasome system can have severe consequences on biological homeostasis. Indeed, mutations that affect the degradation of many of the proteins listed above have been associated with tumorigenesis.
The 26S-proteasome comprises two distinct sub-complexes. The core complex has a sedimentation velocity of 20S and contains a variety of degradative activities. The 20S core is highly conserved across evolutionary distance and consists of a barrel of 4 rings. Each ring contains 7 subunits of either .alpha. class or .alpha. class. The rings are oriented so that two .alpha.-subunit-containing rings are on the outside, while two .beta.-subunit containing rings are juxtaposed on the inside. Thus, the 20S core is identical at its two ends. The x-ray structure of the archaebacterial proteasome has recently been resolved and was shown to contain a narrow pore in each .alpha. ring, and a large central cavity formed by the .beta. rings. Accordingly, the central cavity is not exposed to the cellular environment, thereby preventing non-specific degradation of cellular proteins. Proteins targeted for degradation are first threaded through the narrow pores in the a rings before they gain access to the central catalytic cavity.
The second sub-complex, referred to as the 19S-regulatory complex, binds to the ends of the 20S core and regulates access of cellular proteins to the catalytic cavity. The 19S complex, together with the 20S core make up the 26S-proteasome. The 19S complex has at least 6 distinct ATPase subunits which are thought to promote unfolding of proteolytic substrates so that they can be channeled through the narrow pores of the 20S core. The 19S complex contains as many as 20 subunits, which include a multiubiquitin-chain binding protein, isopeptidases and at least 6 ATPases. To date, many of these additional subunits remain uncharacterized.
The Rad23 gene of S. cerevisiae is necessary for efficient nucleotide excision repair of damaged DNA. In vitro studies indicate that this factor may play a role in assisting the assembly of the repair complex at the site of damage. Accordingly, interactions between Rad23p and other repair proteins including Rad4p, Rad14p, and subunits of TFIIH have been proposed. Thus far, however, the exact biochemical function of Rad23p in DNA repair has remained unclear.
Rad23p has an NH.sub.2 -terminal domain with striking homology to ubiquitin (22% identity, 43% homology). Watkins et al. have shown that this ubiquitin-like domain is required for repair activity of the protein and that the domain can be replaced by the sequence of wild-type ubiquitin. In addition, a family of proteins with similar ubiquitin-like domains have been discovered. Unfortunately, these family members have diverse species of origin and apparently disparate functions and thus have provided no clue as the exact role of this domain.
As noted above, impaired activity of the proteasome is implicated in many diseases in humans. This observation has stimulated considerable research activity in the identification of novel therapeutic agents for inhibiting and/or stimulating the activity of the proteasome. These studies have been hindered by the inefficient, time-consuming, biochemical protocols available for the purification of proteasomes. The present invention describes a rapid and efficient proteasome purification method and provides novel methods of use of various proteasome subunits so purified.